Methods and compositions for increasing longevity and protein yield from a cell culture

ABSTRACT

Disclosed herein are compositions and methods for increasing the longevity of a cell culture and permitting the increased production of proteins, preferably recombinant proteins, such as antibodies, peptides, enzymes, growth factors, interleukins, interferons, hormones, and vaccines. By transfecting cells in culture with an apoptosis-inhibiting gene or vector, cells in culture can survive longer, resulting in extension of the state and yield of protein biosynthesis. Expression of the apoptosis-inhibitor within the cells, because it does not kill the cells, allows the cells, or an increased fraction thereof, to be maintained in culture for longer periods. This invention then allows for controlled, enhanced protein production of cell lines for commercial and research uses, particularly the enhanced production of growth factors, interferons, interleukins, hormones, enzymes, and monoclonal antibodies, and the like. The method preferentially involves eukaryotic cells in culture, and more advantageously mammalian cells in culture.

This application claims priority from U.S. Ser. No. 60/590,349 filed onJul. 23, 2004. This application claims only subject matter disclosed inthe aforementioned provisional application and therefore presents no newmatter.

BACKGROUND OF THE INVENTION

Culturing cells in vitro, especially in large bioreactors, has been thebasis of the production of numerous biotechnology products, and involvesthe elaboration by these cells of protein products into the supportmedium, from which these products are isolated and further processedprior to use clinically. The quantity of protein production over timefrom the cells growing in culture depends on a number of factors, suchas, for example, cell density, cell cycle phase, cellular biosynthesisrates of the proteins, condition of the medium used to support cellviability and growth, and the longevity of the cells in culture (i.e.,how long before they succumb to programmed cell death, or apoptosis).Various methods of improving the viability and lifespan of the cells inculture have been developed, together with methods of increasingproductivity of a desired protein by, for example, controllingnutrients, cell density, oxygen and carbon dioxide content, lactatedehydrogenase, pH, osmolarity, catabolites, etc. For example, increasingcell density can make the process more productive, but can also reducethe lifespan of the cells in culture. Therefore, it may be desirous toreduce the rate of proliferation of such cells in culture when themaximal density is achieved, so as to maintain the cell population inits most productive state as long as possible. This results inincreasing or extending the bioreactor cycle at its production peak,elaborating the desired protein products for a longer period, and thisresults in a higher yield from the bioreactor cycle.

Many different approaches have been pursued to increase the bioreactorcycle time, such as adjusting the medium supporting cell proliferation,addition of certain growth-promoting factors, as well as inhibiting cellproliferation without affecting protein synthesis. One particularapproach aims to increase the lifespan of cultured cells via controllingthe cell cycle by use of genes or antisense oligonucleotides to affectcell cycle targets, whereby a cell is induced into a pseudo-senescencestage by transfecting, transforming, or infecting with a vector thatprevents cell cycle progression and induces a so-called pseudo-senescentstate that blocks further cell division and expands the proteinsynthesis capacity of the cells in culture; in other words, thepseudo-senescent state can be induced by transforming the cells with avector expressing a cell cycle inhibitor (Bucciarelli et al., US Patent2002/0160450 A1; idem., WO 02/16590 A2). The latter method, byinhibiting cell duplication, seeks to force cells into a state that mayhave prolonged cell culture lifetimes, as described by Goldstein andSingal (Exp Cell Res 88, 359-64, 1974; Brenner et al., Oncogene17:199-205, 1998), and may be resistant to apoptosis (Chang et al., ProcNatl Acad Sci USA 97, 4291-6, 2000; Javeland et al., Oncogene 19, 61-8,2000).

Still another approach involves establishing primary, diploid humancells or their derivatives with unlimited proliferation followingtransfection with the adenovirus E1 genes. The new cell lines, one ofwhich is PER.C6 (ECACC deposit number 96022940), which expressesfunctional Ad5 E1A and E1B gene products, can produce recombinantadenoviruses, as well as other viruses (e.g., influenza, herpes simplex,rotavirus, measles) designed for gene therapy and vaccines, as well asfor the production of recombinant therapeutic proteins, such as humangrowth factors and human antibodies (Vogels et al., WO 02/40665 A2).

Other approaches have focused on the use of caspase inhibitors forpreventing or delaying apoptosis in cells. See, for example, U.S. Pat.No. 6,586,206. Still other approaches have tried to use apoptosisinhibitors such as members of the Bcl-2 family for preventing ordelaying apoptosis in cells. See Arden et al., Bioprocessing Journal,3:23-28 (2004). These approaches have yielded unpredictable results; forexample, in one study, expression of Bcl-2 increased cell viability butdid not increase protein production. See Tey et al. Biotechnol. Bioeng.68:31-43 (2000). Another example described overexpression of Bcl-2proteins to delay apoptosis in CHO cells, but Bcl-xL increased proteinproduction whereas Bcl-2 decreased protein production (see WO03/083093).A further example described experiments using expression of Bcl-2proteins to prolong the survival of Sp2/0-Ag14 (ATCC # CRL-1581,hereafter referred to as Sp2/0) cells in cultures; however, the celldensity of the Bcl-2 expressing clones were 20 to 50% lower than that oftheir parental cultures, raising concerns for their practicalapplication in biopharmaceutical industry (see WO03/040374).

It is apparent, therefore, that improved host cells for high levelexpression of recombinant proteins and methods for reliably increasingrecombinant protein production, in particular the production ofantibodies and antibody fragments, multispecific antibodies, fragmentsand single-chain constructs, peptides, enzymes, growth factors,hormones, interleukins, interferons, and vaccines, in host cells aregreatly to be desired.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provideimproved host cells and methods to increase the longevity andrecombinant protein yields of a cell culture by introducing into thecells agents that inhibit senescence or that promote cell survival,e.g., anti-apoptotic agents. The use of such agents preferentiallyincreases the lifespan and viability of cells in culture used for theproduction of a desired recombinant protein, concomitantly increasingthe productivity of such cells in culture, and thereby the optimal yieldof the desired protein. Preferably, the apoptosis inhibitors used in themethod of the present invention include but are not limited to Bcl-2 andits family members. Alternately, the longevity and recombinant proteinyields of a cell clone can be improved by introducing into the cellagents that down-regulate the level of intracellular pro-apoptoticproteins, such as p53 and Rb, or up-regulate intracellularanti-apoptotic proteins, such as Bcl-2. Preferably, the regulatoryagents used in the method of the present invention include, but are notlimited to, human papillomavirus type 16 (HPV-16) oncoproteins E6 andE7, and combinations thereof. Additionally, caspase inhibitors, asdescribed herein, may also contribute to blocking or reducing apoptosis,thus increasing cell survival and increasing the production ofrecombinant proteins by said cells in culture. A further class ofanti-apoptotic agents that can be used in these cultures to enhanceproduction of recombinant proteins includes certain members of thecytokine type I superfamily, such as erythropoietin (EPO). EPO, as aprototype molecule of this class, is a major modifier of apoptosis ofmultiple cell types, not just erythrocytes, and thus has more generalcytoprotective functions, such as in endothelial cells, myocardialcells, tubular epithelial cells of the kidney, skin, and neurons [cf.review by P. Ghezzi and M. Brines, Cell Death and Differentiation 11(suppl. 1), s37-s44, July 2004].

The present invention also teaches cell culture methods incorporatingnovel combinations of factors including, but not limited to,transfection vectors, screening and selection of cell clones withdesired properties, cell culture media, growth conditions, bioreactorconfigurations, and cell types to create cell culture conditions inwhich the longevity of the cell culture is increased and/or made optimaland the yield of a desired recombinant protein is increased. These cellculture methods include suspension, perfusion, and fed-batch methods ofproduction. See Tey et al., J. Biotechnol. 79: 147-159 (2000); Zhang etal., J. Chem. Technol. Biotechnol. 79: 171-181 (2004); Zhou et al.,Biotechnol. Bioeng. 55: 783-792 (1997).

Unless otherwise defined, all technical and scientific terms used in theinvention have the same meaning as commonly understood by one ofordinary skill in the art. In addition, the contents of all patents andother references cited herein are incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF DRAWINGS/FIGURES

FIG. 1 shows visual images of Sp2/0 and Sp-E26 cells treated withcycloheximide (+CHX) or untreated (−CHX).

FIG. 2 shows the results of screening HPV E6/E7 transduced cells thatare more resistant to CHX treatment. A total of 55 clones were screened;in the first experiment, 31 clones were screened (top panel); in thesecond experiment, 24 clones were screened (bottom panel). Healthy cellsof each clone were split into two equal portions. One was treated withCHX for 2 h and the other left untreated. The viable cells in these twocultures were then measured by MTT assay and the ratios of viable cellpopulations treated (CHX⁺) vs. untreated (CHX⁻) were plotted. As shownin the top panel, CHX treatment resulted in 30% reduction of viabilityin Sp2/0 cells, while only 6% reduction in Sp-E26 cells. Seven of the 31clones screened (indicated by *) performed significantly better (<20%reduction of viability) than Sp2/0 but not as well as Sp-E26. For the 24clones screened in the second experiment (bottom panel), CHX treatmentresulted in ˜50% reduction of viability in Sp2/0 cells and <20%reduction of viability in Sp-E26. Ten of the 24 clones (indicated by *or **) screened performed significantly better (<30% reduction) thanSp2/0, and 6 of them (indicated by **) matched or were better thanSp-E26 (<20%). E28 and E36 are two additional control clones thatperform better than Sp2/0 but not as well as Sp-E26.

FIG. 3 shows the dot plots of Guava Nexin V assay. The percentage ofearly apoptotic cells (Nexin V-positive and 7-AAD-negative) is indicatedin the lower-right quadrant.

FIG. 4 shows the DNA fragmentation in Sp2/0 treated by CHX. In contrast,Sp-E26 cells are resistant to the treatment.

FIG. 5 shows the growth profiles of Sp2/0 and Sp-E26 cells in T-flasks.Healthy cells (>95% viability) were seeded in T-flasks at an initialcell density of 200,000/ml. Viable and dead cells were counted dailyusing Guava ViaCount reagent (Guava technologies, Inc.) and PCAinstrumentation (Guava Technologies, Inc.). Accumulation of NH₄ ⁺ andlactate also was monitored.

FIG. 6 compares the growth profiles of Sp2/0 and Sp-E26 cells asdetermined for a batch culture in 3-L bioreactors. Healthy cells (>95%viability) were seeded in the bioreactors at an initial cell density of250,000/ml. Cells were counted daily by trypan blue and microscope.

FIG. 7 shows a representative immunoblot stained with Bcl-2 (100)antibody (Santa Cruz Biotech.) and developed with enhancedchemiluminescence for screening of clones for Bcl-2-EEE expression.

FIG. 8 shows a graph of flow cytometry results using Guava Express.Cells were fixed and permeabilized before staining withphycoerythrin-conjugated anti-Bcl-2 antibody (Santa Cruz Biotechnology,Inc.) Several sub-clones are compared.

FIG. 9 shows a graph of flow cytometry results using Guava Express.Cells were fixed and permeabilized before staining with phycoerythrinconjugated anti-Bcl-2 antibody (Santa Cruz Biotechnology, Inc.). Sp2/0,Raji and Daudi cells were compared to Bcl-2-EEE clones.

FIG. 10 shows the results of immunoblot analyses of 665.B4.1C1, Sp2/0,Raji, Daudi, Sp-EEE (87-29 clone) and Sp-EEE (7-16 clone) cell lysates.A. Blots stained with a human Bcl-2 specific antibody (Santa CruzBiotechnology, Inc). B. Blot stained with an anti-Bcl-2 antibody (SantaCruz Biotechnology, Inc) that recognizes mouse and human Bcl-2.

FIG. 11 shows growth curves (A) and viability (B) of Sp-EEE clonescompared to Sp2/0 cells grown in media supplemented with 10% fetalbovine serum.

FIG. 12 shows growth curves (A) and viability (B) of Sp-EEE clonescompared to Sp2/0 cells grown in media supplemented with 1% fetal bovineserum.

FIG. 13 shows growth curves (A) and viability (B) of Sp-EEE clonescompared to Sp2/0 cells grown in serum-free media.

FIG. 14 shows methotrexate kill curves for Sp-EEE (87-29 clone) cells.

FIG. 15 shows a graph of flow cytometry results using Guava Expresscomparing Sp-EEE clones grown in the presence or absence of 1 mg/mlzeocin. Cells were fixed and permeabilized before staining withphycoerythrin conjugated anti-Bcl-2 antibody (Santa Cruz Biotechnology,Inc).

FIG. 16 shows the map of the pdHL2 vector used to transfect Sp2/0 cellsto obtain the 665.2B9 clone with humanized antibody sequences and theSV40 promoter and enhancer sequences.

FIG. 17 shows the map of DNA plasmid with incorporated Bcl-2 gene, usedfor transfection of clone 665.2B9

FIGS. 18 and 19 show the growth profiles of Bcl-2 transfected clones665.2B9#4, Bcl-2 negative clones and untransfected control. Healthycells (>95% viability) were seeded in 24-well plates at an initial celldensity of 400,000/ml. Viable and dead cells were counted daily usingGuava ViaCount reagent and PCA instrumentation.

FIGS. 20 and 21 show growth profiles of Bcl-2 transfected clone665.259#4 and Bcl-2 negative clones in different MTX concentration.Healthy cells (>95% viability) were seeded in T-flasks at initial celldensity of 100,000/ml. Viable cell density and viability were counteddaily using Guava ViaCount reagent and PCA instrumentation.

FIG. 22 shows the levels of human Bcl-2 expressed by clone 665.2B9#4 inincreasing concentrations of MTX and clone #13 detected by Westernblotting.

FIGS. 23 and 24 show the profiles of cell viability and viable celldensity, respectively, of clone 665.2B9#4 cultured in 0.6 and 1 μM ofMTX and the Bcl-2-negative clone #13 cultured in 0.3 μM MTX with orwithout spiking L-glutamine and glucose. Healthy cells (>95% viability)were seeded in roller bottles at an initial cell density of 200,000/ml.On day 2 and 4 (arrows indicated), a nutrient supplement solutioncontaining glucose and L-glutamine was added to the “spiked” culture.Viable and dead cells were counted daily using Guava ViaCount reagentand PCA instrumentation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides improved compositions, including hostcell lines, and methods for enhanced production of recombinant proteinsin such cell lines. Cell lines have been created that constitutivelyexpress one or more anti-apoptotic genes and that can be transfectedwith an expression construct encoding a protein or peptide of interest,where expression of the anti-apoptotic gene(s) prolongs survival of thetransfected cell in culture and provides for enhanced yields of theprotein or peptide of interest.

Specifically, the present inventors have created from Sp2/0 myeloma cellline two novel cell lines, referred to as Sp-E26 and Sp-EEE, which showenhanced survival in batch culture. Sp-E26 and Sp-EEE constitutivelyexpress the E6 and E7 proteins of HPV-16 and a Bcl-2 mutant, referred toas Bcl-2-EEE, respectively. In addition, recombinant protein production,and particularly production of recombinant antibodies and antibodyfragments, can be improved upon transfecting either Sp-E26 or Sp-EEEwith an expression vector for the recombinant protein of interest. TheE6/E7 or Bcl-2-EEE proteins delay induction of apoptosis in the hostcells and permit enhanced recombinant protein production in the hostcells. Protein production can be boosted still further by addition ofone or more caspase inhibitors (e.g., caspase 1 and/or 3 inhibitors)(Bin Yang et al. Nephron Experimental Nephrology 2004;96:e39-e51),and/or by addition of one or more members of the cytokine type Isuperfamily, such as erythropoietin (EPO), into the growth medium of thecells. A pan-caspase inhibitor is particularly effective in this regard.

The present inventors also have found that production of recombinantproteins, such as antibodies or antibody fragments, can be significantlyenhanced in the host cell by co-expression of an apoptosis inhibitor,such as Bcl-2. In particular, protein production is significantlyenhanced in a myeloma cell line, such as Sp2/0, that is stablytransfected with an expression vector encoding an antibody or antibodyfragment and that is co-transfected with an expression vector encodingan apoptosis inhibitor, such as Bcl-2. Increased production of antibodycan also be obtained from a host cell transfected with the E6/E7 gene.Recombinant protein production can be boosted still further by additionof one or more caspase inhibitors into the growth medium of the cells. Apan-caspase inhibitor is particularly effective in this regard. Also,recombinant protein production can be enhanced by feeding EPO, oranother anti-apoptotic cytokine, into the medium of the cell culture.

Physiological, or programmed, cell death, referred to as apoptosis (Kerret al., Br J Cancer., 26:239-257, 1972) is essential for proper tissuedevelopment and maintenance and is controlled by an intrinsic geneticprogram that has been conserved in evolution (Ellis et al., Annu RevCell Biol, 7, 663-698, 1991). Hence, when cells grow in artificialenvironments, such as ex vivo cultures, this genetic endowment resultsin a finite lifespan. Therefore, the utility of such cell cultures forthe production of proteins used in medicine and industry, as well asresearch, is dependent on maintaining such cultures for extendedlifespan, or cycles, before they die according to apoptotic mechanisms.

Methods and agents have been discovered that act independently on cellproliferation and cell death events, by differentiating cell cycle fromapoptotic effects. Bcl-2, a well-known intracellular regulator ofapoptosis (Vaux et al., Nature 335, 440-2, 1988), is a proto-oncogenethat has been found to have an anti-apototic effect that is geneticallydifferent from its inhibitory influence on cell cycle entry (Huang etal., EMBO J 16, 4628-38, 1997). Two homologues of Bcl-2, Bcl-x_(L) andBcl-w, also extend cell survival, but other members of the Bcl-2 family,such as Bax and Bak, are pro-apoptotic (Oltvai et al., Cell 74, 609-19,1993; Chittenden et al., Nature 374, 733-6, 1995; Farrow et al., Nature374, 731-3, 1995; Kiefer et al., Nature 374, 736-9, 1995). Otheranti-apoptotic genes include Bcl-6 and Mcl-1.

Thus, Bcl-2 and certain of its family members exert protection againstapoptosis, and it was therefore hypothesized as a method to increase thelifespan of certain host cells in culture that are used for theproduction of proteins, thereby enhancing the amount of proteinsproduced and isolated. Since antibodies are produced by B-lymphocytes,particularly by myeloma cells, over-expression of an anti-apoptoticBcl-2 family member, such as Bcl-2, Bcl-x_(L,) Bcl-w or mutant varietiesof these proteins, inhibits apoptosis, resulting in increased celldensity and longer culture survival. Hence, transfection ofanti-apoptotic Bcl-2 family genes avoids the necessity to prolong thecell culture by interfering with the cell cycle per se, as others haveproposed (ibid.). Similarly, transfection of fibroblasts with genes forBcl-2 results in over-expression of Bcl-2 in these cells, resultingagain in an antagonism of apoptosis and increasing the lifespan of thesecells, with a concomitant increase in the production and isolation ofrecombinant proteins. It has also been observed that upon cytokinewithdrawal, interleukin-6 (IL-6)-dependent murine myeloma cells expireas if they undergo apoptosis. It was also found that IL-6-receptors insuch cells could be regulated by Bcl-2 or Bcl-xL in extending apoptosis(Schwarz et al., Cancer Res 55:2262-5, 1995).

Recent literature has also demonstrated that a mutant Bcl-2 possessingthree point mutations (T69E, S70E and S87E) exhibited significantly moreanti-apoptotic activity compared to wild type or single point mutants(Deng et al., PNAS (101) 153-158, 2004). Thus, the invention teaches theconstruction of an expression vector for a Bcl-2-EEE triple mutant,which was then used to transfect Sp2/0 cells to create Sp-EEE clones andsubclones that show improved longevity and recombinant proteinproduction.

Other agents, such as oncogenic viruses, can also oppose apoptosis aspart of their eliciting cellular immortalization and ultimately completemalignant transformation, such as high-risk type HPV oncoproteins E6 andE7 (Finzer et al., Cancer Lett 188, 15-24, 2002). For example, the viralE6 protein effectively blocks the epidermal apoptotic response toultraviolet light (Storey, Trends Mol Med 8, 417-21, 2002). It has alsobeen suggested, from indirect evidence, that the human papillomavirusmay cause reduced apoptosis in squamous (but not basal cell) carcinoma(Jackson et al., Br J Cancer 87, 319-23, 2002). However, not allpapillomavirus oncoproteins have anti-apoptotic effect. For example,other studies have reported that the papillomavirus E6 protein of bovinespecies sensitizes cells to apoptosis (Liu et al., Virology 295, 230-7,2002), which is in contrast to other studies showing that HPV-16 E7 geneprotects astrocytes against apoptosis induced by certain stimuli (Lee etal., Yonsei Med J 42, 471-9, 2001). By use of E6-binding peptideaptamers, direct experimental evidence was obtained that HPV E6oncoprotein has anti-apoptotic activity in HPV-positive tumor cells(Butz et al., Proc Natl Acad Sci USA 97, 6693-7, 2000). However, otherHPV oncoproteins can have the opposite effect; the E2 protein inducesapoptosis in the absence of other HPV proteins (Webster et al., J BiolChem 275, 87-94, 2000). Continuous expression of both the E6 and E7proteins is known to be required for optimal proliferation of cervicalcancer cells and that the two viral proteins exert distinct effects oncell survival (DeFilippis et al., J Virol 77, 1551-63, 2003). Theprimary intracellular target attributed to HPV-16 E6 is p53. E6 forms aternary complex with p53 and a cellular ubiquitin ligase, E6AP,resulting in the ubiquitination and degradation of p53 through theproteosome pathway and inactivation of p53. On the other hand, HPV-16 E7protein interacts and destabilizes the tumor suppressor protein Rb.Moreover, levels of a variety of other intracellular proteins involvedin apoptosis and cell cycle pathways were reported to be regulated by E6and E7 transformation, such as Bcl-2, Bcl-xL, p73, MDM2, p21, cyclinsand cdc, cdk proteins, etc. Changes in the expression of these proteinswill greatly influence the physiological properties of the cell. Thepresent inventors therefore hypothesized that transfection of cells inculture by HPV-16E6 and E7 would be very effective in generatinggenetically modified clones that are resistant toaging-culture-condition induced apoptosis and, therefore, prolong thelifespan of the cell culture. It was also postulated that introductioninto a cell of either HPV-16 oncoprotein E7 or E6 alone might besufficient to generate genetically modified clones with improvedresistance to aging-culture-condition induced apoptosis. When the cellis a recombinant protein-producing clone, the improved physiologicalproperties would in turn translate into enhanced overall proteinproductivity.

Generation of New Host Cells Expressing Viral Anti-Apoptotic Genes

Host cells, such as myeloma host cells, can be generated thatconstitutively express viral anti-apoptotic genes, such as HPV-16 E6 andE7 proteins. These host cells can be transfected with an expressionvector that encodes a recombinant protein of interest and co-expressionof the anti-apoptotic genes results in significantly increasedproduction of the recombinant protein.

The host cell can be essentially any host cell suitable for recombinantprotein production that can be stably transformed with the viralanti-apoptosis genes. For many recombinant proteins, host cells such asCHO and COS cells are advantageous, while for other proteins, such asantibodies, host cells such as myeloma cells and CHO cells are thecommon choices. The viral genes can be introduced into the host cell byany suitable method that results in constitutive or inducible expressionof the genes, i.e., any method that permits stable integration of thegenes into the host cell chromosome while permitting expression of thegenes. Methods for stable transformation of host cells with a gene ofinterest are well known in the art. A particularly advantageous methodis to use a retroviral vector that encodes the viral anti-apoptosisgenes. Suitable vectors include the LSXN vector (Miller et al.Biotechniques 7, 980-90, 1989).

Advantageously, the vector used to transfect the host cell contains aselectable marker that permits selection of cells containing the vector.Suitable selection markers, such as enzymes that confer antibioticresistance on transfected cells, are well known in the art. Aftertransfection, cells are maintained in a medium containing the selectionagent, such as an antibiotic, and screened for resistance to the marker.Cells can be selected and cloned by limiting dilution using conventionalmethods.

The ability of the viral anti-apoptosis genes to increase cell viabilitycan be tested by challenging the cells with an agent that inducesapoptosis, such as cycloheximide (CHX). Cells that do not express theviral anti-apoptosis genes tend to demonstrate significant onset ofapoptosis, whereas cells expressing the genes exhibit drasticallyreduced apoptotic activity. Methods of detecting apoptosis are wellknown in the art and include, for example, cell surface FITC-Annexin Vbinding assay, DNA laddering assay and TUNEL assay.

Upon selection of suitable cells expressing the viral anti-apoptosisgenes, the cells can be transfected with an expression vector encodingthe recombinant protein of choice. The expression vector can be a vectorsuitable for transient expression or, advantageously, can be an episomalvector containing a eukaryotic origin of replication, or an amplifiablevector that permits stable integration and subsequent gene amplificationof the expression cassette. Suitable vectors are well known in the artand include, for example, the pdHL2 vector, which is particularly suitedfor production of antibodies and antibody fragments. When an amplifiableexpression cassette is used, it advantageously contains a selectablemarker that is different from the selectable marker used in theretroviral vector, to allow selection of transfected cells. Once again,suitably transfected cells can be selected and then cloned by limitingdilution.

Upon selection of suitable clones, the cells can be placed in a suitablemedium and cultured to produce the desired protein of interest. Themedium can contain serum or, preferably, be serum-free. In addition,cell longevity and protein production also can be increased by addingone or more caspase inhibitors (e.g., caspase 1 or 3) to the culturemedium. Preferably the caspase inhibitor acts to inhibit one or more ofcaspase 3, caspase 9 and/or caspase 12. A cell-penetrating caspaseinhibitor advantageously is used, and a pan-caspase inhibitor isparticularly advantageous. Suitable inhibitors such as Z-VAD-fmk andAc-DEVD-cho (SEQ ID NO: 7) are well known in the art. Alternatively, thecell line can be further transfected to express a caspase inhibitor,such as Aven or XIAP, to enhance its growth properties by affectingapoptosis. In this regard, certain members of the cytokine type Isuperfamily, such as EPO, can also increase cell survival by havinganti-apoptotic and cytoprotective actions.

The methods described above generate a cell line that can be used fortransfection with essentially any desired gene. However, the skilledartisan will recognize that established cell lines that constitutivelyexpress a desired protein, and particularly a recombinant protein, canbe subsequently transformed with a suitable vector encoding the viral orBcl-2 family anti-apoptosis genes. See Example 2 below.

The protein of interest can be essentially any protein that can beproduced in detectable quantities in the host cell. Examples includetraditional IgG type antibodies, F(ab′).sub.2 or Fab fragments, scFv,diabody, IgG-scFv or Fab-scFv fusion antibodies, IgG- or Fab-peptidetoxin fusion proteins, or vaccines [e.g., including not limited to,Hepatitis A, B or C; HIV, influenza viruses, respiratory syncytialvirus, papilloma viruses, Herpes viruses, Hantaan virus, Ebola viruses,Rota virus, Cytomegalovirus, Leishmania RNA viruses, SARS, malaria,tuberculosis (Mycobacteria), Anthrax, Smallpox, Tularemia, and others].The host cells described herein are particularly suitable for highlyefficient production of antibodies and antibody fragments in myelomacell lines as described in Examples 1 and 2, as well as recombinantgrowth factors (e.g., EPO, G-CSF, GM-CSF, EGF, VEGF, thrombopoietin),hormones, interleukins (e.g., IL-1 through IL-31), interferons (e.g.,alpha, beta, gamma, and consensus), and enzymes. These methods could beapplied to any number of cell lines that are used for production ofrecombinant proteins, including other myeloma cell lines, such as murineNSO or rat YB2/0; epithelial lines, such as CHO and HEK 293; mesenchymalcell lines, such as fibroblast lines COS-1 or COS-7; and neuronal cells,such as retinal cells, as well as glial and glioma cells.

Recombinant Antibody Expression in Cells Expressing Apoptosis Inhibitors

Prior work has described the effects of co-expressing Bcl-2, a naturallyoccurring apoptosis inhibitor, in recombinant CHO cells producing achimeric antibody. See Tey et al., Biotechnol. Bioeng. 68:31-43 (2000).Although increased cell culture life was observed, antibody productiondid not increase over equivalent cells that lacked Bcl-2 expression.However, the present inventors have found that production of recombinantantibody from myeloma cells is significantly increased when the cellsalso express Bcl-2.

Advantageously, the myeloma cell line is stably transfected with anexpression cassette encoding the antibody or antibody fragment. Asuitable expression cassette contains one or more promoters thatcontrols expression of the antibody heavy and light chains (of singlechain in the case of an scFv) together with a selectable marker asdescribed above. A particularly useful vector is pdHL2, which contains aselectable marker gene comprising a promoter operatively linked to a DNAsequence encoding a selectable marker enzyme; a transcription unithaving a promoter operatively linked to a DNA sequence encoding theprotein of interest; an enhancer element between the selectable markergene and the transcription unit, which stimulates transcription of boththe selectable marker gene and the first transcription unit compared tothe transcription of both the selectable marker gene and the firsttranscription unit in the absence of the first enhancer. The vector alsocontains a blocking element having a promoter placed between the firstenhancer and the selectable marker gene, which selectively attenuatesthe stimulation of transcription of the selectable marker gene. V_(H)and V_(L) sequences can be ligated into pdHL2, which is an amplifiablevector containing sequences for the human light chain constant region,the heavy chain constant region, and an amplifiable dhfr gene, eachcontrolled by separate promoters. See Leung et al., Tumor Targeting2:184, (1996) and Losman et al., Cancer 80:2660-2667, (1997). Thisvector can be transfected into cells by, for example, electroporation.Selection can be performed by the addition of 0.1 μM or a suitableconcentration of methotrexate (MTX) into the culture media.Amplification can be carried out in a stepwise fashion with increasingconcentration of MTX, up to 3 μM or higher. Cells stably transfectedwith the expression cassette and that constitutively express theantibody of interest can therefore be obtained and characterized usingmethods that are well known in the art. See also Example 4, below. Afterselection and cloning, the antibody-expressing cell line can then betransfected with an expression vector that encodes an anti-apoptosisgene, such as Bcl-2. For example, the vector pZeoSV (Invitrogen,Carlsbad, Calif.) containing the Bcl-2 gene fused to an SV40 promoter istransfected into the cell using a suitable method such aselectroporation, and selection and gene amplification can be carried outif necessary. Antibody production using the resulting cell line can becarried out as above and compared to production in cells that do notexpress an apoptosis inhibitor.

The methods describe initial preparation of a cell line expressing anantibody or antibody fragment that is subsequently transfected with avector expressing Bcl-2 or a similar inhibitor. However, the skilledartisan will recognize that cell lines can be established thatconstitutively express Bcl-2 or another anti-apoptotic protein, whichcan be subsequently transformed with a suitable vector encoding theantibody or antibody fragment.

Representative examples to illustrate the present invention are givenbelow. Example 1 describes the incorporation of HPV-16 E6/E7 into Sp2/0cell leads to an improved cell clone, Sp-E26, showing characteristics ofreduced/delayed apoptosis. Example 2 describes a method to improve hostcell lines by over-expression of the HPV-16 E7 element alone. Example 3describes using the improved cell, Sp-E26, as a host to develop cellclones producing a recombinant Ab. Example 4 describes the enhancedproduction of Mab observed for an antibody-producing cell line thatco-expresses the E6/E7 element. Example 5 describes the generation andcharacterization of a modified Sp2/0 cell line that constitutivelyexpresses a mutant Bcl-2 (Bcl-2-EEE) possessing three point mutations,resulting in improved longevity. Example 6 describes the improved growthproperties of an antibody-producing cell line that expresses Bcl-2.Example 7 describes the enhanced production of MAb observed for theBcl-2 expressing cell line of Example 6. Example 8 describes the methodsto improve a cell clone producing low-level recombinant protein byintroduction of Bcl-2 expression in the cell. Example 9 describes themethods to improve Sp-E26 by introduction of Bcl-2 expression in thecell. Example 10 describes using the improved cell line, Sp-EEE, as ahost to develop cell clones producing a recombinant Ab. Example 11describes the use of fed-batch reactor profiles and feeding schedules tooptimize yield.

EXAMPLE 1 Generation of Apoptosis-Resistance Cell Clones by StableExpression of HPV-16 E6 and E7 Genes

Selection of Cell Clones Resistant to CHX Treatment

Sp2/0 cells were transduced with an LXSN retroviral vector containingthe expression cassette of HPV-16 E6 and E7 genes at an MOI (multiple ofinfection) of 10:1. After recovery for 24 h, the infected cells wereselected in G418 (1000 μg/ml) for 10 days. G418-resistant cells werecloned in 96-well cell culture plates by limiting dilution (0.5cells/well). Stable infectants were screened for resistance to treatmentby cycloheximide (CHX), a potent apoptosis-inducing agent. Briefly,healthy cells (viability >95%, FIGS. 1C and D) were incubated in mediumcontaining 25 μg/ml of CHX and cell morphology was examined under amicroscope. While more than 50% of parent Sp2/0 cells underwentmorphology change after two to three hours of incubation and becamefragmented (FIG. 1A), several E6/E7 transfected clones showed lessextent of morphology change, indicating resistance to apoptosis. Thebest clone, designated as Sp-E26, showed no apparent morphology changeupon four hours of treatment (FIG. 1B).

To avoid tedious visual examination, MTT assay was used to access thechanges in viable cell population. After the healthy cells wereincubated with or without CHX under normal culture condition for 2-3 h,MTT dye was added to the wells. After further incubation for two hours,the cells were solubilized by adding a lysis buffer contain SDS and HCl.The plates were incubated overnight at 37° C. and OD reading wasperformed at 590 nm using an ELISA plate reader. As shown in FIG. 2, theviable cell population was significantly reduced when Sp2/0 cells weretreated with CHX. By comparison, under the same treatment conditions(concentration of CHX and length of time), Sp-E26 cells tolerated betteragainst CHX treatment. With this method, a large number of clones can bescreened and selected for further analyses (FIG. 2).

Anti-Apoptosis Property of Sp-E26.

CHX-induced apoptosis in Sp-E26 and the parent Sp2/0 cells was evaluatedby Annexin V staining and DNA fragmentation assay. After being incubatedin the medium containing 25 μg/ml of CHX, the cells were harvested andstained with Guava Nexin reagent (equivalent of Annexin V staining) andanalyzed in a Guava Personal Cell Analysis system (Guava Technologies,Inc.). FIG. 3 shows that while more than 30% of Sp2/0 cells becameAnnexin V positive when exposed to CHX treatment for about 1.5 h,indication of apoptosis, Sp-E26 remained healthy, showing no increase inearly apoptotic cells.

The induction of apoptosis by CHX can be revealed by analysis of theformation of intracellular oligonucleosomal DNA fragments, a hallmark ofapoptosis. The cellular DNA was extracted from CHX-treated and untreatedSp-E26 and Sp2/0 cells and DNA laddering assay was performed. In Sp2/0cells treated with CHX, extensive DNA fragmentation was detected (FIG.4). In contrast, under identical treatment conditions, the genomic DNAof Sp-E26 was still intact, showing no appearance of DNA fragmentation(FIG. 4).

Presence of HPV E6 and E7 Genes in Sp-E26

To confirm that E6 and E7 genes are stably present in the genome ofSp-E26 cells, oligonucleotide primers specific for E6 and E7 genes weredesigned and used in a PCR reaction with the genomic DNA extracted fromSp-E26 as the template, resulting in a ˜700 bp DNA fragment. The PCRproduct was cloned and confirmed to be E6 and E7 genes by DNAsequencing. No E6 and E7 genes were detected in the parent Sp2/0 cells.

Improved Growth Properties of Sp-E26

The growth properties of Sp-E26 were evaluated in T-flask (FIG. 5) and3L-batch bioreactor (FIG. 6). Sp-E26 showed improved growth propertiesover the parent Sp2/0 cell in batch cultures, achieving higher maximumcell density and longer survival time.

EXAMPLE 2 Generation of Apoptosis-Resistance Cell Clones by StableOver-Expression of HPV16 E7 Gene

The structure of the poly-cistronic HPV 16 E6 and E7 genes integratedinto the genome of clone Sp-E26 was analyzed by PCR using the primerpair E6-N8⁺(5′-ATG TTT CAG GAC CCA CAG GAG CGA-3′; SEQ ID NO: 8) andE7-C8⁻(5′-TTA TGG TTT CTG AGA ACA GAT GGG-3′; SEQ ID NO: 9) and DNAsequencing. Since the sequences of primer E6-N8⁺ and E7-C8⁻ match withthe coding sequence for the N-terminal 8 amino acid residues of E6 andthe complement sequence for the C-terminal 8 codons of E7, respectively,the amplicon of full-length E6 and E7 is expected to be ˜850 bp.However, amplification of the genomic DNA prepared from Sp-E26 cell withE6-N8⁺ and E7-C8⁻ resulted a PCR fragment of only ˜700 bp. DNAsequencing of the 700 bp PCR product revealed a deletion of a 182poly-nucleotide fragment from the E6 gene. The defective E6 gene islikely resulted from splicing and encodes a truncated E6 peptide withN-terminal 43 amino acid residues. Considering the major physiologicalactivity attributed to E6 is its ability to down-regulate p53expression, the truncated E6 protein is probably not fully functionalbecause the level of p53 expression in Sp-E26 was found to be morestable than that in Sp2/0.

Thus, to evaluate whether HPV-16 E7 gene alone is sufficient to haveanti-apoptotic effect and to improve the growth properties of Sp2/0cells, transfection of Sp2/0 cell with HPV-16 E7 is performed asfollows:

-   (i) The DNA sequence encoding E7 is cloned from Sp-E26 cell by    RT-PCR. Proper restriction sites are introduced to facilitate the    ligation of the gene into a mammalian expression vector, pRc/CMV    (Invitrogen). Transcription of the viral gene within the vector,    designated as E7pRc, is directed from CMV promoter-enhancer    sequences. The vector also contains a gene conferring neomycin    resistance, which is transcribed from the SV40 promoter.-   (ii) Sp2/0 cells are transfected with the expression vector    containing the expression cassette of HPV-16 E7 gene. Briefly, 5 ug    of E7pRc is linearized by ScaI and transfected into the cell by    electroporation.-   (iii) After recovery for 24 hours, the transfected cells are    selected in G418 (1000 μg/ml) for 10 days.-   (iv) G418-resistant cells are then cloned in 96-well cell culture    plates by limiting dilution (0.5 cells/well). Stable transfectants    are selected and screened for resistance to treatment by    cycloheximide (CHX), a potent apoptosis-inducing agent.-   (v) Healthy cells (viability >95%) are incubated in medium    containing 25 μg/ml of CHX or in the absence of CHX for 3-4 hours    under normal culture conditions, followed by the addition of MTT dye    into the wells. After further incubation for two hours, the cells    are solubilized by adding a lysis buffer contain SDS and HCl. The    plates are incubated overnight at 37° C. and an OD reading is    performed at 590 nm using an ELISA plate reader. Cell clones showing    resistance to CHX treatment are selected and expanded for further    analyses.-   (vi) The anti-apoptosis property of E7-transfected cells is    evaluated by Annexin V staining and DNA fragmentation assays. In the    Annexin V assay, after being incubated in the medium containing 25    μg/ml of CHX, the cells are harvested and stained with Guava Nexin    reagent (equivalent of Annexin V staining) and analyzed in a Guava    Personal Cell Analysis system (Guava Technologies, Inc.). In the DNA    fragmentation assay, the cellular DNA is extracted from CHX-treated    and untreated E7-transfectants and Sp2/0 cells and analyzed with    agarose gel electrophoresis.-   (vii) Expression of the viral oncogene in E7-transfectants is    evaluated by Southern blot (genomic level), Northern blot (mRNA    level), and immunoblot (protein level) analysis. Expression of    intracellular proteins that are involved in apoptosis processes and    affected by E7 protein are examined by immunoblotting analyses.-   (viii) The growth properties of selected E7-transfectants are    evaluated in T-flask and in a 3L-batch bioreactor. The transfectants    show improved growth properties, i.e. achieving higher maximum cell    density and longer survival time, over the parent Sp2/0 cell in    batch cultures are considered to be better host cells.

EXAMPLE 3 High-Level Expression of hLL2 IgG in Sp-E26

In this example, Sp-E26 is used as a host to generate cell clonesproducing hLL2, a humanized anti-CD22 Ab developed for treating patientswith NHL and autoimmune diseases. An hLL2-producing clone, 87-2-C9, waspreviously generated by using Sp2/0 cell as a host (Losman et al.,Cancer 80, 2660-2666, 1997), in which case, only one positive clone (afrequency of ˜2.5×10⁻⁷) was identified after transfection, and themaximum productivity (P_(max)), defined as the concentration of theantibody in conditioned terminal culture medium in T-flask, of the onlyhLL2-producing clone, before amplification, was 1.4 mg/L. Transfectionof Sp-E26 cell with the same hLL2pdHL2 vector and by using similarprocedures as described by Losman et al. (Cancer 80, 2660-2666, 1997)resulted in more than 200 stable hLL2-producing clones, a frequency of>10⁻⁴). The P_(max) of 12 randomly selected clones was evaluated andfound to be between 13 and 170 mg/L, with a mean of 50 mg/L. Theproductivities of these clones can be further enhanced by geneamplification with MTX. This example demonstrated the advantage of usingSp-E26 over its parent Sp2/0 cell as a host for the development of cellclones producing recombinant proteins.

EXAMPLE 4 Improvement of Ab-Producing Cell Lines by Stable Expression ofHPV16 E6 and E7 Genes

607-3u-8 cells were originally generated from Sp2/0 by transfection toproduce a humanized monoclonal Ab. The clone was developed by geneamplification (with MTX) and subcloning to enhance the maximum (Ab)productivity up to 150 mg/L, which decreased to ˜100 mg/L followingweaning off serum supplement in the culture medium. To obtain higherantibody productivity under serum-free condition, E6/E7 genes of HPV-16were introduced into 607-3u-8 and the effect of E6/E7 on Ab-productivitywas evaluated as follows. 607-3u-8 cells maintained in HSFM supplementedwith 10% FBS and 3 uM MTX were transduced with an LXSN retroviral vectorcontaining the expression cassette of HPV-16 E6 and E7 genes at an MOIof 10:1. After recovery for 24 h, stably transfected cells were selectedin G418 (400 μg/ml) for 10 days. G418-resistant cells were subcloned in96-well cell culture plates by limiting dilution (0.5 cells/well). Asurviving clone, designated as 607E1C12, was obtained for evaluation.Two subclones, designated as 607-3u-8-7G7 and 607-3u-8-2D10, of 607-3u-8without E6/E7 transfection were also selected. The P_(max) of thesethree clones were determined and there were no significant difference(Table 1). These results suggest that introducing E6/E7 genes into thecell does not alter the ability of cells producing Ab. Next, 607E1C12,607-3u-8-7G7 and 607-3u-8-2D10 were adapted to grow in serum-free mediumand the productivities of these clones were determined. All cells weregrowing well in serum-free medium. The final antibody productivity ofclone 607E1C12 was maintained at 150 mg/L, while the two clones withoutE6/E7 were substantially reduced. In addition, the productivity of607E1C12 were stable after a freeze (for cryopreservation) and thawcycle (Table 1)

TABLE 1 The productivities of Ab-producing clones P_(max)(mg/L)^(a)Clone With serum Serum-free 607-3u-8-7G7 127 ± 16 (3)^(b)  74 ± 10 (4)607-3u-8-2D10 140 ± 4 (3)   35 ± 2 (2) 607E1C12 154 (1) 142 ± 13 (6)607E1C12 (Cryo)^(c) 145 ± 17 (5) ^(a)Determined by protein purificationof IgG from terminal culture supernatants. ^(b)The number in parenthesisindicates the sample size. ^(c)Cells had been frozen forcryopreservation.

EXAMPLE 5 Generation and Characterization of a Genetically ModifiedSp2/0 Cell Line that Constitutively Expresses a Mutant Bcl-2

Evidence suggests that a mutant Bcl-2 possessing three point mutations(T69E, S70E and S87E) exhibits significantly more anti-apoptoticactivity compared to wild type or single point mutants (Deng et al.,PNAS 101: 153-158, 2004). Thus, an expression vector for this triplemutant (designated as Bcl-2-EEE) was constructed and used to transfectSp2/0 cells for increased survival and productivity, particularly inbioreactors. Clones were isolated and evaluated for Bcl-2-EEE expressionlevel, growth and apoptotic properties. The nucleic acid sequence forthe Bcl-2-EEE is depicted as SEQ. ID. No. 3; the corresponding aminoacid sequence for the Bcl-2-EEE protein is depicted as SEQ. ID. No. 4.

A 116 bp synthetic DNA duplex was designed based on the coding sequencefor amino acid residues 64-101 of human Bcl-2. The codons for residues69, 70 and 87 were all changed to those for glutamic acid (E). Theentire sequence was extraordinarily GC rich and had numerous poly G andpoly C runs. Conservative changes were made to several codons to breakup the G and C runs and decrease the overall GC content.

Two 80-mer oligonucleotides were synthesized that, combined, span the116 bp sequence and overlap on their 3′ ends with 22 bp (See SEQ. ID.No. 5 & 6). The oligonucleotides were annealed and duplex DNA wasgenerated by primer extension with Taq DNA polymerase. The duplex wasamplified using the PCR primers, Bcl-2-EEE PCR Left(5′-TATATGGACCCGGTCGCCAGAGAAG-3′; SEQ ID NO: 10), and Bcl-2-EEE PCRRight (5′-TTAATCGCCGGCCTGGCGGAGGGTC-3′; SEQ ID NO: 11).

The 126 bp amplimer was then cloned into pGemT PCR cloning vector. TheBcl-2-EEE-pGemT construct was digested with TthI and NgoMI restrictionendonucleases and the 105 bp fragment was gel isolated and ligated withhBcl-2-puc19 vector (ATCC 79804) that was digested with Tthl and NgoMIto generate hBcl-2(EEE)-puc19. The sequence of this construct wasconfirmed.

A 948 bp insert fragment was excised from hBcl-2 (EEE)-puc19 with EcoRIand ligated with pZeoSV2+ vector that was digested with EcoRI andtreated with alkaline phosphatase. The resulting construct is hBcl-2(EEE)-pZeoSV2+.

Sp2/0 cells (5.6×10⁶) were then transfected by electroporation with 60μg of hBcl-2 (EEE)-pZeoSV2+ following the standard protocol for Sp2/0cells. The cells were plated into six 96-well plates that were incubatedwithout selection for 48 hours. After two days, 800 μg/ml of zeocin wasadded to the media.

Cells from 40 wells were expanded to 24-well plates and analyzed bywestern blot with anti-hBcl-2 and anti-β actin. All but 5 of the 40showed medium to high levels of Bcl-2-EEE expression. The results forone of four gels are shown in FIG. 7. An Sp2/0 derived hMN 14 cell line(Clone 664.B4) that was previously transfected with wild type Bcl-2 wasused as a positive control (+). As was demonstrated by Deng et al., theBcl-2-EEE migrates slightly slower than wild type Bcl-2 in SDS-PAGE.

Three strongly positive wells (#7, #25 and #87) were chosen for furtherevaluation and sub-cloning. Limiting dilution plating resulted in <20positive wells per 96-well plate, indicating a very high probability(>99%) that the cells in individual wells are in fact cloned. Initially,23 subclones from the three original wells were analyzed by GuavaExpress using anti-hBcl-2-PE (FIG. 8). The results confirmed that theoriginal wells contained mixed cell clones. Well #7 yielded clones withthe strongest signal and well #25 had those with the lowest. Clones7-12, 7-16, 87-2 and 87-10 were expanded for further analysis.Subsequently, some initially slower growing subclones were similarlyanalyzed and one clone, 87-29, gave a signal that was 20% higher thanany other clone and was expanded for further analysis. Two highexpressing SP-EEE clones (87-29 and 7-16) were compared to theuntransfected Sp2/0, Raji and Daudi cells (FIG. 9). The Sp-EEE clonesexpresses about 20-fold higher than Raji and Daudi cells, which are bothknown to express Bcl-2 at presumably normal cell levels. Sp2/0 cellswere negative. This was further verified by anti-Bcl-2 immunoblot (FIG.10). Bcl-2 was not detected with a human Bcl-2 specific antibody inSp2/0 cells even with high protein loading (50K cells) and long exposureof X-ray film. Immunoblot analysis with an anti-Bcl-2 MAb (C-2, SantaCruz Biotech.) that recognizes mouse, rat and human Bcl-2 did not detectany Bcl-2 from untransfected Sp2/0 cells, even with high protein loading(100K cells) and long exposure time (FIG. 10B). If there is any Bcl-2expressed in Sp2/0 cells, it is at a level that is more than 2 orders ofmagnitude less than the Bcl-2-EEE in clone 87-29. Growth curves werecompared for five Sp-EEE subclones and Sp2/0 cells. Three Sp-EEEsubclones displayed a clear advantage over Sp2/0 cells. These three(7-12, 7-16 and 87-29) also express the highest levels of Bcl-2-EEE. As7-12 and 7-16 are from the same original well and have nearly identicalproperties (Bcl-2-EEE levels and growth curves), they likely originatedfrom the same initial clone. The best two SP-EEE subclones 7-16 and87-29 were used for further evaluation.

The clones were plated in media supplemented with 10%, 1% or 0% serum(without weaning) and cell density and viability were monitored. In 10%serum 87-29 grew to a high density and had more than 4 days increasedsurvival compared to Sp2/0 cells (FIG. 11). In 1% serum, all cells grewto about 35-40% of the density achieved in 10% serum and the Bcl-2-EEEtransfectants had a similar survival advantage over Sp2/0 (FIG. 12).When transferred directly into serum free media, the Sp2/0 cells onlygrew to 600K cells/ml while 87-29 cells grew to a two-fold higherdensity (FIG. 13). In each serum concentration 87-29 cells survived 4-6days longer than Sp2/0 cells.

The methotrexate (MTX) sensitivity was determined for 87-29 (FIG. 14).The data suggests that a minimum MTX concentration of 0.04 μM issufficient for initial selection of MTX-resistant clones. Therefore, thesame selection and amplification protocols used for Sp2/0 cells can beemployed with the SP-EEE cells.

Bcl-2 is a pro-survival/anti-apoptotic protein. It has been demonstratedby several groups that a Bcl-2 deletion mutant missing the flexible loopdomain (FLD) has an enhanced ability to inhibit apoptotosis (Figueroa etal., 2001, Biotechnology and Bioengineering, 73, 211-222; Chang et al.,1997, EMBO J., 16, 968-977). More recently, it was demonstrated thatmutation of 1 to 3 S/T residues in the FLD of Bcl-2 to glutamic acid,which mimics phosphorylation, significantly enhances its anti-apoptoticability (Deng et al. 2004, PNAS, 101, 153-158). The triple mutant (T69E,S70E and S87E) provided the most significant survival enhancement. Here,the present invention teaches the generation of a similar Bcl-2 triplemutant construct (Bcl-2-EEE), which is used to stably transfect Sp2/0cells.

All the aforementioned experiments demonstrate that expression ofBcl-2-EEE reduces apoptosis rates in Sp2/0 cells. This effect waslargely dose dependent, in that clones with higher expression levelssurvived longer than those with lower levels. The best clone, 87-29,grows to a 15-20% higher cell density and survives an additional 4-6days compared to untransfected Sp2/0 cells.

The Bcl-2-EEE level in clone (87-29) is approximately 20-fold higherthan normal levels in Daudi or Raji cells. No Bcl-2 expression wasdetected in untransfected Sp2/0 cells. As described in Example 6,hMN-14-expressing Sp2/0 cells were transfected with a similar constructfor expression of wild type Bcl-2 and a clone with exceptional growthproperties and enhanced productivity was isolated. When this clone(664.B4) was amplified further with MTX, the Bcl-2 levels increasedsignificantly. Ultimately, the amplified (3 μM MTX) cell line wassub-cloned and the Bcl-2 level of one clone (664.B4.1C1) was two-foldhigher than 664.B4. This particular subclone has superior productivityand growth properties. The Bcl-2-EEE level in 87-29 is approximatelytwo-fold higher than the level of Bcl-2 in the amplified 664.B4.1C1.87-29 cells have a growth rate that is comparable to that of Sp2/0 cellsand can apparently continue to grow for one additional day and reach amaximal density that is 15-20% higher than Sp2/0. A similar property wasfound for the E6/E7 expressing Sp-E26 cell line. The Bcl-2-EEEexpressing 87-29 clone, which provides an additional 4-6 days survivalover the parental Sp2/0 cells, is superior to the Sp-E26 clone, whichonly survives one additional day.

The Sp-EEE cell line as represented by the 87-29 clone is useful as anapoptosis-resistant host for expressing a recombinant protein upontransfection with a suitable vector containing the gene for thatrecombinant protein. In order for this cell line to be useful it mustmaintain its Bcl-2-EEE expression and survival advantage followingtransfection and amplification and during extended culture. It isunlikely that the stably transfected Bcl-2-EEE gene will be lost duringsubsequent transfection and therefore the survival properties should notdiminish. It is possible that MTX amplification could even improve thesurvival of producing clones via increasing expression of Bcl-2proteins. Indeed, this was the case with the hMN-14 664.B4 cell line,which was transfected with wild type Bcl-2. Following amplification andsub-cloning, the Bcl-2 level increased several fold and cell survivalimproved significantly.

EXAMPLE 6 Improvement of Ab-Producing Cell Survival in Stationary BatchCulture by Stable Expression of a Human Bcl-2 Gene

Generation of a Bcl-2-Transfected Cell Clone

A cell clone 665.2B9 was originally generated from Sp2/0 by transfectionto produce a humanized monoclonal anti-CEA Ab (Qu et al., unpublishedresults). A vector, designated hMN14pdHL2, was used to transfect Sp2/0cells to obtain the cell clone 665.2B9. The pdHL2 vector was firstdescribed by Gillies et al., and had an amplifiable murine dhfr genethat allows subsequent selection and amplification by methotrexatetreatment (Gillies et al., J. Immunol. Methods 125:191 (1989)).Generally, the pdHL2 vector provides expression of both IgG heavy andlight chain genes that are independently controlled by twometallothionine promoters and IgH enhancers. A diagram of the hMN14pdHL2vector is shown in FIG. 16. SEQ. ID. No. 1 shows the sequence of thevector; SEQ. ID. No. 2 shows the 72 bp sequence defined as the enhancersequence; the promoter sequence corresponds to nt2908-2979 ofhMN14pdHL2.

Sp2/0 cells can be generally transfected by electroporation withlinearized pdHL2 vectors such as the hMN14pdHL2 vector used in thisinstance. Selection can be initiated 48 hours after transfection byincubating cells with medium containing 0.05 to 0.1 μM MTX.Amplification of inserted antibody sequences is achieved by a stepwiseincrease in MTX concentration up to 5 μM.

The clone was subjected to gene amplification with MTX increasedstepwise to 0.3 μM, at which point the maximum productivity (Pmax) ofthe antibody was increased to about 100 mg/L. To improve cell growthproperties, 665.2B9 cells were transfected with a plasmid expressionvector (FIG. 17) containing the human Bcl-2 gene by electroporation.Bcl-2 gene was excised from pB4 plasmid purchased from ATCC (pB4,catalog #79804) using EcoRI sites and inserted into MCS of mammalianexpression vector pZeoSV(+) using the same restriction enzyme. Sincezeocin resistance gene is part of the vector, transfected cells wereplaced into medium containing zeocin ranging from 50-300 μg/mL. Stableclones were selected from media containing 300 mg/ml zeocin andsubcloned in media without zeocin by plating into 96-well plates at adensity of 0.5 cell/100 uL/well. The media without zeocin was usedthereafter. Formation of clones in wells was confirmed by visualobservation under a microscope. Cells from the wells with only 1 clusterof cells were expanded. Each 96-well plate produced around 30 clones,from which 14 clones were randomly selected for further studies. Thegrowth characteristics of these clones were evaluated by daily cellcounting and viability measurements with ViaCount reagent and Guava PCA.From the 14 clones evaluated in 24-well plates (FIGS. 18, 19), oneBcl-2-transfected clone showing improved growth characteristics (highercell densities and prolonged cell survival) was identified anddesignated as 665.2B9#4 (or clone #4). Comparing to the parent 665.2B9clone, clone #4 grew to a higher cell density (˜1.7-fold) and survived 4to 6 days longer in T-flasks (FIGS. 20, 21), and as a consequence ofbetter growth, the P_(max) of clone #4 was increased to about 170 mg/Las determined by ELISA titration and Protein A column purification.

Bcl-2 Expression in 665.2B9#4

To confirm that the improved growth properties of 665.2B9#4 wereresulted from transfection of Bcl-2, intracellular level of human Bcl-2protein was measured by using Guava Express reagent and Guava PCAinstrument. Briefly, 4×10⁵ cells placed in 1.5 ml spin-tubes werecentrifuged for 5 minutes at 1500 rpm, washed three times with 1×PBS.Supernatants were carefully aspirated. Fixation solution (10×, 60 μL)from Santa Cruz Biotechnology (SCB), Inc. (cat. # sc-3622) was added tocell pellets for 15 min and incubated on ice. Fixation solution wasremoved with 4×1 mL PBS at 4° C., each time spinning as described.Permeabilization buffer (0.5 mL) at −20° C. (SCB cat. # sc-3623) wasadded dropwise while vortexing, followed by 15 min incubation on ice.Cells were then spun and washed two times with 0.5 mL FCM wash buffer(SCB cat. # sc-3624). Final cell pellet was resuspended in 100 μL of FCMwash buffer and stained for Bcl-2 intracellular protein with 10 μL ofanti-Bcl-2 mouse monoclonal antibody conjugated to PE (obtained fromSCB). Incubation was performed at room temperature in dark for one hour.Two washes with 0.5 mL of FCM wash buffer followed. The final cellpellet was resuspended with 0.4 mL FCM wash buffer and the cellsanalyzed on Guava PC. Mean values of the fluorescence intensity (MFI)for each clone were compared to control staining with non-specific,isotype mouse IgGI conjugated with PE. The results summarized in Table 2confirm that clone 665.2B9#4 expresses a higher level of Bcl-2 proteincompared to the parental cell line. A zeocin-resistant clone (#13) thatshowed a similar growth profile as the parent 665.2B9 was negative forBcl-2 staining, confirming that Bcl-2 expression is necessary for theimprovement of growth.

TABLE 2 Intracellular level of Bcl-2 determined by Guava Express.Viability^(a) Cell (%) Mean FI (AU) 665.2B9 84 42 665.2B9#4 97 110Clone#13 92 14 Non-specific antibody 12 staining ^(a)Determined beforethe assay to ensure healthy cells were used. ^(b)665.2B9 cells stainedwith an isotype-matched mouse IgG1antibody, PE-conjugated.With Guava Express analysis it was found that the intensities offluorescent staining corresponding to Bcl-2 levels are rising with MTXamplification of clone 665.2B9#4, suggesting co-amplification of Bcl-2with the dhfr gene. To compare intracellular Bcl-2 levels of amplifiedcells, Western blotting analysis was performed on cell lysates of clone665.2B9#4 (Bcl-2 positive) and clone #13 (Bcl-2 negative) using ananti-human Bcl-2 antibody. Densitometric evaluation showed that Bcl-2signal of clone 665.2B9#4 growing in 1.0 μM MTX is 2× stronger than thecells in 0.6 μM MTX. A lysate of Clone #13 did not reveal the presenceof Bcl-2 protein (FIG. 22).

EXAMPLE 7 Improved Ab-Production of Clone 665.2B9#4 Under Batch CultureCondition

By monitoring nutrients consumption in the cell cultures near theterminal phase, it was found that glucose and L-glutamine are the firstcomponents to be consumed. Experiments were carried out to determinewhether supplementation of these limiting nutrients would improve thefinal antibody yields. Two types of cultures were initiated: spiked fedbatch—where these limiting components were supplemented upon theirconsumption; and unfed batch—without nutrients supplementation. Testedwere Bcl-2-positive clone 665.2B9#4 growing in medium containing 0.6 and1 μM of MTX and the Bcl-2-negative clone #13 growing in 0.3 μM MTX.FIGS. 23 and 24 show the profiles of cell viability and cell density inboth culture types until they reached terminal stage. Protein yields,expressed as mg/L, are shown in Table 3. The results of this experimentsuggest that nutrient spiking improves total yield of produced antibodyabout 2-fold for all cultures.

TABLE 3 Antibody production under batch culture conditions Unfedbatch^(a) Spiked fed batch^(a) Cell/MTX (μM) (mg/L) (mg/L) 665.2B9#4/0.6117 286 665.2B9#4/1.0 156^(b) 296 Clone#13/0.3  74.1 165 ^(a)Determinedby Protein A column purification. ^(b)Average of two purifications.

EXAMPLE 8 Introduction of Bcl-2 Gene into a Cell Line ProducingLow-Level of Recombinant Protein

A cell clone 482.2C4A was originally generated from Sp2/0 bytransfection to produce a bispecific Ab in the form of an IgG (anti-CEA)and two scFv (anti-DTPA) (Leung et al., J. Nuc. Med. 41: 270P, 2000;Hayes et al., Proc. Am. Asso. Cancer. Res. 43: 969, 2002), each of whichis covalently linked to the C-terminus of the IgG heavy chain. The clonewas subjected to gene amplification and had a final productivity of ˜20mg/L. To improve the growth property and eventually the Ab productivity,482.2C4A cells were transfected with a plasmid expression vectorcontaining the human Bcl-2 gene by electroporation as described inExample 6. The transfectants were selected in medium containing 750μg/ml of Zeocin after three weeks.

Zeocin-resistant cells were treated with 25 μg/ml of CHX for 5 hours toeliminate apoptosis-sensitive cells. Treated cells were washed twicewith fresh culture medium to remove CHX and resuspended in fresh growthmedium. After recovering for 24 h, the viable cells were cloned into96-well cell culture plates by limiting dilution (0.5 cells/well).Clones emerged in the wells in two weeks and were screened for Abproduction, resistance to CHX-induced apoptosis, as well as growthprofiles. Those clones performed better than the parent 482.2C4A in allaspects are selected and further characterized. The best performer isexpected to be more robust when growing under stress condition, resistto aging-culture-condition induced apoptosis, and have a higher maximumAb productivity (ca. 150% or better) comparing to the parent 482.2C4Acell.

EXAMPLE 9 Introduction of Bcl-2 Gene into Sp-E26 for a FurtherImprovement of Cell Growth Properties

Sp-E26 cells are transfected with a plasmid expression vector containingthe human Bcl-2-EEE gene, as described in Example 5, by electroporation.The transfectants are selected in medium containing 500 μg/ml of Zeocinafter three weeks.

Zeocin-resistant cells are treated with 25 μg/ml of CHX for 5 hours toeliminate apoptosis-sensitive cells. Treated cells are washed twice withfresh culture medium to remove CHX and resuspended in fresh growthmedium. After recovering for 24 h, the viable cells are cloned into96-well cell culture plates by limiting dilution (0.5 cells/well).Clones emerge in the wells in two weeks and are screened for resistanceto CHX-induced apoptosis, as well as growth profiles. Those clonesperform better than the parent Sp-E26, as well as Sp-EEE, in all aspectsare selected and further characterized. The best performer containingHPV-16 E6/E7 and Bcl-2-EEE is expected to be more robust when growingunder stress condition and resistant to aging-culture-condition-inducedapoptosis than the parent Sp-E26 and Sp/EEE cells; therefore, it is abetter host cell for recombinant protein production.

EXAMPLE 10 Improved Production of Recombinant Proteins with the Sp-EEECell Line

There are two paths that can be taken when developing a cell line withenhanced survival for production of recombinant proteins. One method,which has been accomplished quite successfully, as described in Example6, involves stable transfection of an already producing cell line with apro-survival gene, such as Bcl-2. However, this method requiresadditional transfection, selection and cloning steps, therebylengthening the cell line development process by at least two months andpossibly much more. Further, screening for the “best” clone is ratherinvolved, since a number of parameters need to be determined for eachclone, including growth/survival, Bcl-2 expression level andproductivity. Thus, only a small number of clones can be evaluated. Itis quite possible that clones with the highest productivity may not havesuperior survival and vice versa. An alternative strategy, employedhere, is to develop a parental cell line with superior growth andsurvival properties, which is subsequently transfected with theexpression vector for production of the desired protein.

Compared to Sp2/0 cells, the Sp-EEE cells continue to grow for oneadditional day, reach a maximal density that is 15-20% higher, andsurvive an additional 4-6 days in culture. The cells retain theirenhanced growth and survival properties when subsequently transfectedwith genes for the production of recombinant proteins, such as IgG,antibody fragments and fusion proteins, growth factors, such as G-CSF,GM-CFS, EPO, EGF, VEGF, cytokines, such as an interleukin family member(IL-1-IL-31), or interferon family members (such as alpha, beta or gammainterferon), oligonucleotides, peptides, hormones, enzymes, or vaccines(e.g., Hepatitis A, B or C, as well as others described above).

A DNA vector, such as pdHL2, containing one or more expression cassettesfor recombinant protein(s), such as an IgG, is used to transfect Sp-EEEcells by standard methods, such as electroporation. The transfectantsare plated in 96-well plates and clones are analyzed for proteinproduction by established techniques such as ELISA or Biacore.Productive clones are subjected to increasing concentrations of MTX inthe culture media over several months to amplify the genetic copynumber. Since the Bcl-2-EEE-expressing clones grow to ˜20% higher celldensity and survive at least an additional 4 days as compared to clonesgenerated in Bcl-2 negative Sp2/0 cells, the former will produce atleast 20% more recombinant protein in standard flask or roller bottleculture. An even greater increase is realized in suspension, perfusionor fed-batch bioreactor cultures.

EXAMPLE 11 Improved Ab-Production of Bcl-2 Transfected Clone 665.B4.1C1in Bioreactor

Both 665.2B9#4 and the parent clone 665.2B9 of Example 6 were weanedinto serum-free media. The cells were adapted to a customizedformulation of hybridoma serum-free medium (HSFM) (Immunomedics PN10070) containing 3 μM MTX by continuous subculture in T-flasks forseveral months. The adapted cells were scaled up from T-flasks to rollerbottles for banking. A master cell bank (MCB) for each cell line wascreated with 1×10⁷ viable cells in each 1-mL vial using an FBS-freecryopreservation solution composed of 45% conditioned medium (mediumthat is collected as supernatant after centrifugation of a culture inthe exponential growth phase), 10% DMSO and 45% HSFM. The MCB cell lineswere designated 665.2B9.1E4 (without Bcl-2 gene) and 665.B4.1C1 (withBcl-2 gene), respectively. The growth properties and antibody productionof these two clones were compared under batch culture conditions.

Experiments were conducted in 3-L bench-scale bioreactors using theabove cells expanded from the MCB. The 3-L bioreactor system is thescale-down model of a 2500-L cGMP bioreactor system. Therefore, theevaluation results would support the suitability of these cell lines forlarge-scale commercial manufacturing.

The same growth HSFM as that used in creating the MCB (Immunomedics PN10070) was used to maintain the cell line and prepare the inoculum.Basal HSFM, a customized formulation based on the growth HSFM withcustomized modifications (Immunomedics PN 10194), was used in the 3-Lfed-batch bioreactor process. Both media contain insulin and transferrinas the only trace proteins. Additional 0.1% Pluronic F68 wasincorporated into the formulation to protect cells from shear caused byagitation and aeration. This media also contained 3 μM MTX.

The specific characteristics of the continuous feeding solutions and thepulse feeding solutions are shown in tables 4 and 5 as follows:

TABLE 4 Continuous feeding solutions Solutions Formulation (Dissolve inWFI unless specified) Glucose and glutamine Glucose, 13.3 g/L;Glutamine, 20 mM solution (G/G) ImmuC2 solution Glucose, 13.3 g/L;Glutamine, 20 mM; PNS A, 50 ml/L; NaOH, 50 mM ImmuC5 solution Glucose,26.6 g/L; Glutamine, 40 mM; PNS A, 100 ml/L; NaOH, 100 mM

TABLE 5 Pulse feeding Solutions Formulation Solutions (Dissolve in WFIunless specified) TC Soy Plus 120 g/L Linoleic acid/cyclodextrin 1.5mg/ml HD lipid 500X β-mercaptoethanol/EDTA BME, 0.01 M; EDTA, 0.1 mM.ImmuVitamin MEM Vitamin Solution (100x), as solvent; Choline Chloride,500 mg/L; Myo-inositol, 600 mg/L. TEC solution Transferrin solution (4mg/mL), as solvent; CaCl2, 125 mM; Ethanolamine-HCl 1 g/L. Insulin 4mg/ml

The fed-batch experiments were conducted in 3 L Bellco spinner-flaskbioreactor systems (Bellco glasses, Vineland, N.J.) with 2 L of workingvolume. The bioreactor temperature, pH and dissolved oxygen (DO) weremonitored and controlled by single loop controllers. The reactortemperature was controlled at 37° C. by a heating blanket. The culturepH was controlled at 7.3 by the addition of CO₂ or 6% Na₂CO₃. Aerationwas performed through a cylindrical sintered sparger at 10 ml/min. DOwas controlled above 40% of air saturation by intermittent sparging ofO₂ into the medium. A constant agitation rate of 50˜60 rpm was usedthroughout the cultivation.

A frozen vial from MCB was thawed and recovered in T-flasks inapproximately 1 to 2 weeks. The cells were then expanded from T-flasksto roller bottles prior to inoculation into the bioreactors. Cells werecultured at 37° C. in a 5% CO₂ atmosphere and maintained in theexponential growth phase throughout the expansion process.

Prior to the inoculation, 1.2 liters of Basal HSFM was pump-transferredinto the bioreactor aseptically. The medium was air saturated tocalibrate the dissolved oxygen (DO) probe. A medium sample was alsotaken to calibrate the pH probe. Once pH probes and DO probes werecalibrated, both controllers were set to AUTO modes. Once the systemreached set points of pH (7.3) and temperature (37° C.), calculatedamount of inoculum from roller bottle was pump transferred into thebioreactor. The post-inoculation viable cell density (VCD) was around2×10⁵ vial cells/ml.

The feeding strategy is as follows. During the cultivation, concentratednutrient solutions were fed into the bioreactor to provide the cellswith necessary and non-excessive nutrients. Concentrated nutrientsolutions were delivered to the culture via continuous feeding and pulsefeeding. The continuous feeding solutions were pump transferred into thereactor continuously using peristaltic pumps (Watson-Marlow 101U/R). Thepulse feeding solutions were pulse fed once a day into the culture.

Two fed-batch feeding strategies were developed and applied to both celllines. Process #1 does not feed recombinant insulin during thecultivation. Process #2 is designed based on Process #1 with a modifiedlinoleic acid and lipid feeding schedule and an additional feeding ofinsulin.

The following tables summarize the feedings of both processes for bothcell lines.

TABLE 6 Process #1 for cell line 665.2B9.1E4 Continuous feedingContinuous Feeding Rate (ml/day) Expected Viable Cell Glucose and DayDensity (cells/mL) Glutamine ImmuC2 ImmuC5 Day 2  0.4~0.7E6 60 0 0 Day 3am  1.0~1.7E6 0 60 0 Day 3 pm 1.01~2.5E6 0 90 0 Day 4 am 2.51~3.5E6 0 900 Day 4 pm 2.51~4.5E6 0 150 0 Day 5 am 4.51~6.5E6 0 0 90 Day 5 pm4.51~7.5E6 0 0 120 Day 6 7.51~12E6  0 0 120 Day 7 if <13E6 0 0 120if >13.1E6 0 0 150 Pulse feeding Pulse Feeding (mL) TC Soy Plus LA/CDLipid TEC Immu BME/ Day (120 g/L) (1.5 mg/ml) (500X) Solution VitaminEDTA Day 3 12.5 4 3 — — 15 Day 4 25 8 — — — — Day 5 50 12  — 4 15 15 Day6 60 8 2 8 — — Day 7 60 — 1 — — — Day 8 25

TABLE 7 Process #2 for cell line 665.2B9.1E4 Continuous feeding ExpectedViable Cell Continuous Feeding Rate (ml/day) Day Density (cells/mL)Glucose and Glutamine ImmuC2 ImmuC5 Day 2  0.4~0.7E6 60 0 0 Day 3 am 1.0~1.7E6 0 60 0 Day 3 pm 1.01~2.5E6 0 90 0 Day 4 am 2.51~3.5E6 0 90 0Day 4 pm 2.51~4.5E6 0 150 0 Day 5 am 4.51~6.5E6 0 0 90 Day 5 pm4.51~7.5E6 0 0 120 Day 6 7.51~12E6  0 0 120 Day 7 if <13E6 0 0 120if >13.1E6 0 0 150 Day8 if <10E6 0 0 90 if 10.1~13E6    0 0 120if >13.1E6 0 0 150 Pulse feeding Pulse Feeding (mL) TC Soy Plus LA/CDLipid TEC Immu BME/ Insulin Day (120 g/L) (1.5 mg/ml) (500X) SolutionVitamin EDTA (4 mg/ml) Day 3 12.5 2 3 — — 15 — Day 4 25 4 — — — — — Day5 50 6 — 4 15 15 4 Day 6 60 4 — 8 — — 8 Day 7 60 4 — — — 15 8 Day 8 50 —— — — — 8

TABLE 8 Process #1 for cell line 665.B4.1C1 Continuous feeding ExpectedContinuous Feeding Rate (ml/day) Viable Cell Glucose and Day Density(cells/mL) Glutamine ImmuC2 ImmuC5 Day 2  0.4~0.7E6 60 0 0 Day 3 am 1.0~1.7E6 0 90 0 Day 3 pm 1.01~2.5E6 0 120 0 Day 4 am 2.51~3.5E6 0 0 60Day 4 pm 2.51~4.5E6 0 0 90 Day 5 am 4.51~6.5E6 0 0 120 Day 5 pm4.51~7.5E6 0 0 150 Day 6 7.51~12E6  0 0 180 Day 7, 8, 9 if <15E6 0 0 180if >15.1E6 0 0 240 Day10 if <10E6 0 0 120 if 10.1~13E6    0 0 150if >13.1E6 0 0 180 Pulse feeding Pulse Feeding (mL) TC Soy Plus LA/CDLipid TEC Immu BME/ Day (120 g/L) (1.5 mg/ml) (500X) Solution VitaminEDTA Day 3 12.5 4 3 — — 15 Day 4 25 8 — — — — Day 5 50 12  — 4 15 15 Day6 60 8 2 8 — — Day 7 60 — 1 — — 15 Day 8 60 — — — — — Day 9 60 — — — —15 Day 10 50 — — — — —

TABLE 9 Process #2 for cell line 665.B4.1C1 Continuous feeding ExpectedViable Cell Continuous Feeding Rate (ml/day) Day Density (cells/mL)Glucose and Glutamine ImmuC2 ImmuC5 Day 2  0.4~0.7E6 60 0 0 Day 3 am 1.0~1.7E6 0 90 0 Day 3 pm 1.01~2.5E6 0 120 0 Day 4 am 2.51~3.5E6 0 0 60Day 4 pm 2.51~4.5E6 0 0 90 Day 5 am 4.51~6.5E6 0 0 120 Day 5 pm4.51~7.5E6 0 0 150 Day 6 7.51~12E6  0 0 180 Day 7, 8, 9 if <15E6 0 0 180if >15.1E6 0 0 240 Day 10 if <10E6 0 0 120 if 10.1~13E6    0 0 150if >13.1E6 0 0 180 Pulse feeding Pulse Feeding (mL) TC Soy Plus LA/CDLipid TEC Immu BME/ Insulin Day (120 g/L) (1.5 mg/ml) (500X) SolutionVitamin EDTA (4 mg/ml) Day 3 12.5 2 3 — — 15 — Day 4 25 4 — — — — — Day5 50 6 — 4 15 15 4 Day 6 60 4 — 8 — — 8 Day 7 60 4 — — — 15 8 Day 8 60 4— — — — 8 Day 9 60 4 — 4 15 15 8 Day 10 60 — — — —    1− 8

During the cultivation, bioreactor samples were taken periodically foroff-line analysis. The viable cell density (VCD) and the cell viabilitywere measured by microscopic counting using a hemocytometer afterstaining with 0.4% trypan blue dye. The glucose, lactate, glutamine,ammonia concentrations were measured using a Nova Bioprofile 200. Theantibody concentration was determined by HPLC using a protein A affinitychromatography column (Applied Biosystems, P/N 2-1001-00).

The specific antibody productivity was calculated by dividing thecumulative antibody produced by the time integral of the total viablecell in the culture:

${Q_{\lbrack{MAb}\rbrack} = \frac{( {{\lbrack{Mab}\rbrack_{t\; 1} \cdot V_{t\; 1}} - {\lbrack{Mab}\rbrack_{t\; 0} \cdot V_{t\; 0}}} }{\int_{t\; 0}^{t\; 1}{{VCD} \cdot {Vdt}}}},{{in}\mspace{14mu}{which}}$$\int_{t\; 0}^{t\; 1}{{{VCD} \cdot {Vdt}}\mspace{14mu}{is}\mspace{14mu}{approximated}\mspace{14mu}{by}\mspace{14mu}{the}\mspace{14mu}{Trapezium}{\;\mspace{11mu}}{Rule}\text{:}\mspace{14mu}\frac{( {{{VCD}_{t\; 0} \cdot V_{t\; 0}} + {{VCD}_{t\; 1} \cdot V_{t\; 1}}} )( {{t\; 1} - {t\; 0}} )}{2}}$

By Process #1, 665.2B9.1E4 cells grew to attain a maximal VCD of 1×10⁷viable cells/ml on day 6 with 86% of viability. After day 6, VCD and V%decreased quickly and the culture was harvested on day 8. Process #2helped the culture reach a higher VCD of 1.2×10⁷ viable cells/ml andsustain one day longer.

As compared to 665.2B9.1E4 cells, 665.B4.1C1 cells exhibited much bettergrowth in both processes. In Process #1, its VCD reached 2×10⁷ viablecells/ml on day 7 with 97% viability. The culture also maintained thisVCD and V % for two more days before it started to decline. The culturewas harvested on day 11. In Process #2, 665.B4.1C1 cells showed asimilar growth profile as in Processes #1. More specifically, the cellsreached the highest VCD of 2.3×10⁻⁷ viable cells/ml and the viabilitydeclined a little slower with the harvest occurring on day 11. Thisobservation was somewhat different from the 665.2B9.1E4 cell line, whichdemonstrated a growth advantage in Process #2.

The antibody yields of two cell lines in Processes #1 and #2 werecompared. The final yield of 665.2B9.1E4 cells was 0.42 g/L in Process#1 and 0.55 g/L in Process #2. For comparison, 665.B4.1C1 cellsdelivered a higher final yield of 1.5 g/L in both processes.

The daily specific antibody productivities (per cell basis) werecalculated and the 665.2B9.1E4 cells had an average daily Q_([MAb]) ofapproximately 15 pg/cell/day throughout the course of cultivation forboth processes. The additional day of growth at the highest VCD inProcess #2 resulted in a higher final antibody concentration.

The 665.B4.1C1 cells showed a similar daily specific antibodyproductivity profile in both processes with Process #1 yielding slightlyhigher productivity. The daily Q_([MAb]) were maintained between 20˜25pg/cell/day until day 9. Thereafter the productivity declined.

Comparing with the 665.2B9.1E4 cell line, the 665.B4.1C1 cell lineexhibited a higher specific antibody productivity of 20˜25 pg/cell/dayas compared to 15 pg/cell/day. Combining with its better growth, the665.B4.1C1 cell line tripled the final antibody yield to 1.5 g/L ascompared to 0.55 g/L achieved by the 665.2B9.1E4 cell line. Theseresults demonstrate the benefit of incorporating Bcl-2 gene into thehost cell line to enhance its growth and antibody yield in serum-freemedia in a bioreactor modeled for large-scale commercial preparation ofa recombinant protein, in this case an antibody for clinical use. Themethods and processes disclosed herein can be modified, as appropriate,by one skilled in the art. All publications, patents and patentapplications, and references contained therein, are incorporated hereinby reference in their entirety.

1. A cell culture comprising a clonal population of Sp2/0 host cells,the Sp2/0 host cells modified with a first nucleic acid sequencecomprising SEQ ID NO:3, the first nucleic acid sequence integrated inthe host cell chromosomal DNA, the first nucleic acid encoding aheterologous Bcl-EEE protein that inhibits apoptosis, the host cellfurther modified with a second nucleic acid sequence encoding arecombinant protein, the clonal population of host cells in a mediumsuitable for cell growth.
 2. The cell culture according to claim 1,wherein the medium comprises a caspase inhibitor selected from the groupconsisting of Z-VAD-fmk, Ac-DEVD-cho (SEQ ID NO: 7), Aven and XIAP. 3.The cell culture according to claim 1, wherein said medium comprises anexogenously added member of the cytokine type I superfamily.
 4. The cellculture according to claim 3 wherein said member of the cytokine type Isuperfamily is erythropoietin.
 5. The cell culture according to claim 1,wherein said recombinant protein is an antibody or antibody fragment. 6.The cell culture according to claim 5, wherein Sp2/0 host cells modifiedwith a nucleic acid sequence encoding the Bcl-EEE heterologous proteingrow to a higher cell density than unmodified Sp2/0 host cells under thesame culture conditions.
 7. The cell culture according to claim 5,wherein Sp2/0 host cells modified with a nucleic acid sequence encodingthe Bcl-EEE heterologous protein grow to a cell density that is 15 to20% higher than unmodified Sp2/0 host cells under the same cultureconditions.
 8. The cell culture according to claim 5, wherein Sp2/0 hostcells modified with a nucleic acid sequence encoding the Bcl-EEEheterologous protein grow to a cell density that is 35 to 40% higherthan unmodified Sp2/0 host cells when grown in 1% serum medium.
 9. Thecell culture according to claim 5, wherein Sp2/0 host cells modifiedwith a nucleic acid sequence encoding the Bcl-EEE heterologous proteingrow to a two-fold higher cell density in serum-free medium thanunmodified Sp2/0 host cells.
 10. The cell culture according to claim 5,wherein the clonal population of modified Sp2/0 host cells has beenexposed to methotrexate to amplify the nucleic acid sequences.
 11. Thecell culture according to claim 5, wherein the methotrexate exposureresults in a two-fold amplification of the Bcl-EEE encoding firstnucleic acid.
 12. The cell culture according to claim 1, wherein theSp2/0 cells are transfected with a vector comprising the first nucleicacid.
 13. The cell culture according to claim 1, wherein the Sp2/0 cellsare transfected with a vector comprising the second nucleic acid. 14.The cell culture according to claim 12, wherein the vector is the pdHL2vector.
 15. The cell culture according to claim 13, wherein the vectoris the pdHL2 vector.
 16. The cell culture according to claim 5, whereinSp2/0 host cells modified with a nucleic acid sequence encoding theBcl-EEE heterologous protein produce at least 20% more recombinantprotein than Sp2/0 host cells that are not modified with the nucleicacid sequence encoding the Bcl-EEE heterologous protein.
 17. The cellculture according to claim 1, wherein the Sp2/0 cells are transfectedwith the first nucleic acid before they are transfected with the secondnucleic acid.